Meconopsis biluoensis (Papaveraceae), a new species revealed by population‐level investigation

Abstract Meconopsis biluoensis, a new species of Papaveraceae in an alpine meadow from Yunnan, Southwest China, is described and illustrated. Morphologically, it resembles Meconopsis georgei, while it is distinct in acaulescent and hispid with clearly expanded bases on the leaves. A genus‐level molecular phylogenetic analysis supported the closest relationship between M. biluoensis and M. georgei. In a finer population‐level molecular phylogenetic analysis using ribosomal DNA (rDNA) and the chloroplast genome, individuals from M. biluoensis and M. georgei were clearly separated, and the extremely short branch length indicated that the two species had a very short differentiation time. The species has currently been assessed as “endangered” (EN) due to its small‐sized population and narrow distribution following the IUCN categories and criteria.

monitor the species in Nanjieluo and conducted a botanical exploration in Fuchuan Mountain this year, the type locality of M. georgei.
In Fuchuan Mountain, we collected individuals who were nearly the same as those in the protologue of M. georgei morphologically.After carefully comparing the chloroplast genome and nuclear gene rDNA at the population level, we believe that there are stable differences between the individuals in Nanjieluo and Fuchuan Mountain, both morphologically and genomically.Therefore, the individuals found in Nanjieluo should be a new species in Meconopsis.
Total genomic DNA was extracted and purified using the Plant Genomic DNA Rapid Extraction kit (Tiangen Corporation, Beijing, China).The quality of the DNA was evaluated using a NanoDrop spectrophotometer (Thermo Scientific, Carlsbad, CA, USA), while the integrity of the DNA was assessed through electrophoresis on a 1% (w/v) agarose gel.A DNA library was prepared using the NEB Next Ultra DNA Library Prep Kit (Illumina Inc., San Diego, CA, USA).
The DNA libraries were then sequenced on an Illumina NovaSeq 6000 (Illumina Inc.) platform to generate 150-bp paired-end reads, with approximately 5 GB of data for each sample.Low-quality reads from raw data were filtered using fastp v 0.23.1 to remove sequencing adaptors and low-quality bases (Chen et al., 2018).The filtered reads were assembled using the GetOrganelle v1.7.7.0 program for plastomes and nrDNA (Jin et al., 2020).The chloroplast genome sequences were initially annotated using Geneious Prime by referring to the cp genome sequence of Meconopsis punicea (MW233592.1)and subsequently refined through manual calibration based on the open reading frame.The NCBI database accession numbers of the new sequences (cp genomes, nr DNA) in this study and other species for molecular phylogenetic analysis are shown in Table A1.

| RE SULTS
In the genus-level molecular phylogenetic analysis using the single chloroplast gene matK, our result was highly congruent with that of earlier studies (Xiao & Simpson, 2017).In the maximum likelihood tree, four clades were formed.The inferred new species M. biluoensis was embedded in sect Aculeate according to Xiao and Simpson (2017), together with M. georgei.The two species formed a monophyletic group in clade Aculeate (Figure 1).
To further investigate the genetic differentiation between M. biluoensis and M. georgei, we carried out a more detailed molecular phylogenetic study using the chloroplast genome and nuclear rDNA at the population level.In this analysis, M. biluoensis was represented by five individuals, and M. georgei was represented by 13 individuals.
Both the chloroplast genome (Figure 2) and nrDNA tree (Figure 3) of the ML and BI methods show nearly the same topology.Individuals from different species were grouped together separately with the highest support.

| DISCUSS ION
The genus-level molecular phylogenetic tree shows that M. georgei and M. biluoensis formed a monophyletic group and were embedded in section Aculeatea according to Xiao's classification (Xiao & Simpson, 2017).In a further population-level molecular phylogenetic analysis, both the chloroplast genome and nuclear rDNA supported the monophyly of the two species.Our previous results showed that it was not possible to differentiate M. biluoensis from M. georgei using ITS (Wei et al., 2019).With the number of samples increasing, we conducted another molecular phylogenetic analysis using ITS (Figure 4).In the ITS tree, M. georgei forms a monophyletic group, together with a sequence from GenBank (No. JX078989.1),which was amplified from the same gathering as the type specimen of M. georgei collected by Georgei Forrest.Due to poor resolution caused by insufficient information sites, M. biluoensis is paraphyletic apart from M. georgei.However, as the sequence length and information sites increase, the monophyly of M. biluoensis is equally well demonstrated at the biparental genetic marker rDNA sequence (Figure 3).It is worth noting that the branch lengths of M. biluoensis and M. georgei are extremely short, indicating that the differentiation time of these two species is very short.This result may explain the subtle morphological differences between the two species.
Meconopsis is an herbaceous genus native to high-altitude habitats across the Himalayas and adjacent plateau and mountain areas.The Himalo-Tibetan region is the best-known region in the world to be undergoing orogenesis (Van Hinsbergen et al., 2012).
Many studies suggest that mountain uplift can drive the evolution of new species and the rapid differentiation of species (Hughes & Eastwood, 2006;Meng et al., 2007;Winkworth et al., 2005).
Meconopsis may be undergoing these processes.During speciation and rapid differentiation, differences between species may have developed gradually, accompanied by hybridization (De Queiroz, 2007), making morphological features within genera more complex than we can imagine.Therefore, we must be very careful when addressing the nuances between species in Meconopsis.
The main difference between M. biluoensis and M. georgei is the type of inflorescence.Scapes versus racemose is a vital difference discussed by many authors in their monograph of Meconopsis to distinguish sections and series (Grey-Wilson, 2014; Taylor & Cox, 1934;Wu et al., 1999;Zhang & Grey-Wilson, 2008).this difference in 2019.When consulting the type specimens, we noticed that the inflorescence was not a typical racemose, and all flowers were loosely arranged on long, slender pedicels along the elongated stem.It is easy to imagine that when some individuals grow shorter, lax racemose can change into leafless scapes (Wei et al., 2019).This situation occurs in other species in Meconopsis, such as M. horridula and M. lancifolia.However, after 5 years of observation, we found that the difference in inflorescence was fairly stable between the two species (during the 5-year monitoring process, we recorded over 50 flowering plants of M. biluoensis, and all individuals possessed leafless scapes; in contrast, all individuals of M. georgei we saw this year possessed lax racemose [Figure 5]).
Therefore, the type of inflorescence could be an important diagnostic feature between M. biluoensis and M. georgei.
Another difference is the indumenta, especially on the leaves.
In M. biluoensis, the hispid have conspicuous expanded bases, while in M. georgei, hispid scarcely expand at the base, which was also mentioned by Grey-Wilson in his monograph on Meconopsis (Grey-Wilson, 2014).It is worth noting that when only comparing specimens, the difference becomes less insignificant because the expanded base of the hairs collapsed when losing moisture during the process of making specimens.That is the reason we did not take it as a diagnostic feature before.However, when we take a closer observation of plants in their living state, the difference is obvious and stable at the population level.At the same time, we performed a simple scanning electron microscopy observation.In the case of dehydration, whether the base is expanded is still very obvious between the two species (Figure 6).In summary, combining molecular phylogeny and morphological surveys at the population level, we believe M. biluoensis is a separate species with a very short history of differentiation.

| DIAG NOS IS
Meconopsis biluoensis is morphologically similar to M. georgei, but it can be distinguished from the latter in having leafless scapes (vs.lax raceme) and hispid on the leaves with an expanded base (vs.base of hispid scarcely expand).The color of the expanded base is usually brown or black (Figure 9).Additionally, M. biluoensis is usually a dwarf plant, rarely exceeding 20 cm, while M. georgei is usually taller than 20 cm.Detailed comparisons of diagnostic characteristics are listed in Table 1.

| Phenology
Flowering from mid-July to early August and fruiting from August to September.

| Etymology
The species is named after the mountain range to which it was discovered and endemic: Biluo Xueshan Mountain.

| Distribution and ecology
This new species is presently only known from the type locality: Nanjieluo village, Badi township, Biluo Xueshan, Weixi, northwest Yunnan, with an elevation range from 4000 to 4200 m.Meconopsis biluoensis grows in stony and rocky alpine meadows and slopes, where the environment is often wet and mossy.

| Conservation status
During five consecutive years of fieldwork in Biluo Xueshan Mountain, we only found M. biluoensis in Nanjieluo.In this area, we found a total of five populations, with each population comprising approximately 10-30 individuals (Wang et al., 2023).Considering that there are areas in Biluo Xueshan Mountain that are difficult to reach, it is also possible that M. biluoensis could be distributed in suitable habitats in these regions.However, based on the experience of the current survey, the number of individuals per colony will not exceed 50.Therefore, we estimate the total number of this species to be approximately 500.Based on the above surveys and estimates, we determined its endangerment level to be endangered (EN) based on IUCN (2012), quoting clause C, subclause C.2a.

ACK N OWLED G M ENTS
We are grateful to Mr. Bin Wang from Gaoligong Mountain National Nature Reserve Lushui Management and Protection Branch for his help during the field work.Meanwhile, we pay our highest respect to the late Mr. Yoshida for his suggestion about this new species.

FU N D I N G I N FO R M ATI O N
This study is supported by Zhilan Foundation, Shenzhen (Grant No.

CO N FLI C T O F I NTE R E S T S TATE M E NT
There is no conflict of interest to declare.
Flowers of M. biluoensis are born on leafless scapes, while the flowers of M. georgei are arranged into a racemose.We noticed F I G U R E 1 ML tree inferred from matK sequence data.Values below branches are ML bootstrap percentages.Meconopsis biluoensis is shown in red.F I G U R E 2 Population level tree inferred from cp genome focusing only Meconopsis georgei and Meconopsis biluoensis.Values below branches are ML bootstrap percentages and Bayesian posterior probabilities.F I G U R E 3 Population level tree inferred from nrDNA focusing only Meconopsis georgei and Meconopsis biluoensis.Values below branches are ML bootstrap percentages and Bayesian posterior probabilities.

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I G U R E 4 Population-level tree inferred from ITS focusing only Meconopsis georgei and Meconopsis biluoensis.Values below branches are Bayesian posterior probabilities.The orange M. georgei represents the sequence from the same gathering as the type specimen of M. georgei which already exists in the GenBank.

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Comparison between Meconopsis georgei (a) and Meconopsis biluoensis (b), showing the major diagnostic characteristics.TA B L E 1 Diagnostic character between Meconopsis georgei and Meconopsis biluoensis.